Project Summary It is established that the human pathogen Legionella pneumophila becomes significantly augmented for infection of macrophages after intracellular growth in amoebae when compared to like-strains cultivated in laboratory media. Based on this observation, we reasoned that the most critical virulence determinants of L. pneumophila are expressed by responding to stimuli generated by the protozoan host specifically; a process we term ?protozoan-priming?. We sought to identify L. pneumophila virulence factors that were specifically required for replication in amoebae in order to highlight the genes necessary for production of most infectious form of the bacterium. Using a transposon mutagenic screen, we successfully identified twelve insertions that produced bacteria severely attenuated for growth in amoebae, while retaining a functional Dot/Icm type IVb secretion system. Seven of these insertion mutants were found dispensable for growth in macrophages; revealing potential attractive therapeutic targets that reside upstream of the pathogen-human interface. Two candidates identified, lpg0730 and lpg0122 are conserved among numerous important human pathogenic bacteria that can use amoebae as an environmental reservoir. Each gene encodes a component of an ATP- binding cassette (ABC) transport complex of unknown function. We hypothesize that Lpg0730 and Lpg0122- containing complexes comprise purine and sulfonated compound importers, respectively. In this study, we will characterize these proteins in order to determine their contribution to intracellular survival of L. pneumophila, and potentially highlight conserved survival mechanisms employed by bacteria to utilize protozoa as an environmental reservoir for replication.